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• W1569591326 abstract "SummaryBackground: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α2β1. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates αIIbβ3 in platelets and αLβ2 in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α2β1. Methods: Using ADAP−/− mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP−/− platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α2β1‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α2β1, was reduced in ADAP−/− platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP−/− platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α2β1. In addition, we found that ADAP−/− mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α2β1. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates αIIbβ3 in platelets and αLβ2 in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α2β1. Methods: Using ADAP−/− mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP−/− platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α2β1‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α2β1, was reduced in ADAP−/− platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP−/− platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α2β1. In addition, we found that ADAP−/− mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation. Platelet activation by collagen is a key step in the initiation of arterial thrombosis [1Farndale R.W. Sixma J.J. Barnes M.J. De Groot P.G. The role of collagen in thrombosis and hemostasis.J Thromb Haemost. 2004; 2: 561-73Crossref PubMed Scopus (305) Google Scholar]. Collagen interacts directly with platelets via glycoprotein (GP)VI and integrin α2β1: GPVI is the principal collagen signaling receptor on platelets [2Nieswandt B. Watson S.P. Platelet–collagen interaction: is GPVI the central receptor?.Blood. 2003; 102: 449-61Crossref PubMed Scopus (913) Google Scholar], and although α2β1 serves mainly as an adhesion receptor [3Varga‐Szabo D. Pleines I. Nieswandt B. Cell adhesion mechanisms in platelets.Arterioscler Thromb Vasc Biol. 2008; 28: 403-12Crossref PubMed Scopus (454) Google Scholar], several studies have suggested that it may also mediate collagen‐induced signaling [4Ichinohe T. Takayama H. Ezumi Y. Arai M. Yamamoto N. Takahashi H. Okuma M. Collagen‐stimulated activation of Syk but not c‐Src is severely compromised in human platelets lacking membrane glycoprotein VI.J Biol Chem. 1997; 272: 63-8Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 5Jarvis G.E. Best D. Watson S.P. Glycoprotein VI/Fc receptor gamma chain‐independent tyrosine phosphorylation and activation of murine platelets by collagen.Biochem J. 2004; 383: 581-8Crossref PubMed Scopus (7) Google Scholar, 6Inoue O. Suzuki‐Inoue K. Dean W.L. Frampton J. Watson S.P. Integrin α2β1 mediates outside‐in regulation of platelet spreading on collagen through activation of Src kinases and PLCγ2.J Cell Biol. 2003; 160: 769-80Crossref PubMed Scopus (204) Google Scholar, 7Sundaresan P. Farndale R.W. P38 mitogen‐activated protein kinase dephosphorylation is regulated by protein phosphatase 2A in human platelets activated by collagen.FEBS Lett. 2002; 528: 139-44Crossref PubMed Scopus (40) Google Scholar, 8Mazzucato M. Cozzi M.R. Battiston M. Jandrot‐Perrus M. Mongiat M. Marchese P. Kunicki T.J. Ruggeri Z.M. De Marco L. Distinct spatio‐temporal Ca2+ signaling elicited by integrin alpha2beta1 and glycoprotein VI under flow.Blood. 2009; 114: 2793-801Crossref PubMed Scopus (36) Google Scholar]. The response to collagen of GPVI‐deficient platelets is weak, like that of Fc receptor γ‐chain (FcRγ)‐deficient (FcRγ−/−) platelets, which also lack GPVI [9Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. Expression and function of the mouse collagen receptor glycoprotein VI is strictly dependent on its association with the FcRgamma chain.J Biol Chem. 2000; 275: 23998-4002Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 10Moroi M. Jung S.M. Okuma M. Shinmyozu K. A patient with platelets deficient in glycoprotein VI that lack both collagen‐induced aggregation and adhesion.J Clin Invest. 1989; 84: 1440-5Crossref PubMed Scopus (370) Google Scholar]. We have previously shown that collagen induces tyrosine phosphorylation of adhesion and degranulation‐promoting adapter protein (ADAP) in FcRγ−/− platelets [5Jarvis G.E. Best D. Watson S.P. Glycoprotein VI/Fc receptor gamma chain‐independent tyrosine phosphorylation and activation of murine platelets by collagen.Biochem J. 2004; 383: 581-8Crossref PubMed Scopus (7) Google Scholar] to a degree comparable with that induced in wild‐type (WT) platelets by the GPVI‐selective agonist collagen‐related peptide (CRP). Phosphorylation of ADAP by collagen in WT platelets was substantially greater [5Jarvis G.E. Best D. Watson S.P. Glycoprotein VI/Fc receptor gamma chain‐independent tyrosine phosphorylation and activation of murine platelets by collagen.Biochem J. 2004; 383: 581-8Crossref PubMed Scopus (7) Google Scholar]. This suggests that, although collagen can regulate ADAP through GPVI signaling, other pathways may also be important. ADAP is present in both T cells and platelets [11Wang H. Rudd C.E. SKAP‐55, SKAP‐55‐related and ADAP adaptors modulate integrin‐mediated immune‐cell adhesion.Trends Cell Biol. 2008; 18: 486-93Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar]. ADAP participates in the regulation of αLβ2 following T‐cell receptor activation [12Griffiths E.K. Krawczyk C. Kong Y.Y. Raab M. Hyduk S.J. Bouchard D. Chan V.S. Kozieradzki I. Oliveira‐Dos‐Santos A.J. Wakeham A. Ohashi P.S. Cybulsky M.I. Rudd C.E. Penninger J.M. Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/Slap.Science. 2001; 293: 2260-3Crossref PubMed Scopus (248) Google Scholar, 13Wang H. Wei B. Bismuth G. Rudd C.E. SLP‐76‐ADAP adaptor module regulates LFA‐1 mediated costimulation and T cell motility.Proc Natl Acad Sci USA. 2009; 106: 12436-41Crossref PubMed Scopus (62) Google Scholar, 14Peterson E.J. Woods M.L. Dmowski S.A. Derimanov G. Jordan M.S. Wu J.N. Myung P.S. Liu Q.H. Pribila J.T. Freedman B.D. Shimizu Y. Koretzky G.A. Coupling of the TCR to integrin activation by Slap‐130/Fyb.Science. 2001; 293: 2263-5Crossref PubMed Scopus (267) Google Scholar], in the regulation of αIIbβ3 on platelets activated by von Willebrand factor [15Kasirer‐Friede A. Moran B. Nagrampa‐Orje J. Swanson K. Ruggeri Z.M. Schraven B. Neel B.G. Koretzky G. Shattil S.J. ADAP is required for normal alphaIIbbeta3 activation by VWF/GP Ib–IX–V and other agonists.Blood. 2007; 109: 1018-25Crossref PubMed Scopus (53) Google Scholar], and in mechanotransduction induced by shear forces exerted on platelets tethered to αIIbβ3 [16Kasirer‐Friede A. Ruggeri Z.M. Shattil S.J. Role for ADAP in shear flow‐induced platelet mechanotransduction.Blood. 2010; 115: 2274-82Crossref PubMed Scopus (32) Google Scholar]. It has been reported that ADAP‐deficient (ADAP−/−) mice have a mild hemostatic deficit [15Kasirer‐Friede A. Moran B. Nagrampa‐Orje J. Swanson K. Ruggeri Z.M. Schraven B. Neel B.G. Koretzky G. Shattil S.J. ADAP is required for normal alphaIIbbeta3 activation by VWF/GP Ib–IX–V and other agonists.Blood. 2007; 109: 1018-25Crossref PubMed Scopus (53) Google Scholar] and mild thrombocytopenia [14Peterson E.J. Woods M.L. Dmowski S.A. Derimanov G. Jordan M.S. Wu J.N. Myung P.S. Liu Q.H. Pribila J.T. Freedman B.D. Shimizu Y. Koretzky G.A. Coupling of the TCR to integrin activation by Slap‐130/Fyb.Science. 2001; 293: 2263-5Crossref PubMed Scopus (267) Google Scholar]. In this study, we found that ADAP participates in collagen‐induced platelet responses, mediated via both GPVI and, especially, α2β1. FcRγ−/− C57/Bl6 mice were generated by Professor Takashi Saito (Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba, Japan) [17Park S.Y. Ueda S. Ohno H. Hamano Y. Tanaka M. Shiratori T. Yamazaki T. Arase H. Arase N. Karasawa A. Sato S. Ledermann B. Kondo Y. Okumura K. Ra C. Saito T. Resistance of Fc receptor‐deficient mice to fatal glomerulonephritis.J Clin Invest. 1998; 102: 1229-38Crossref PubMed Scopus (239) Google Scholar]; ADAP−/− C57/Bl6 mice were generated by Dr Erik J Peterson (Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA) and Professor Gary A Koretzky (University of Pennsylvania, Philadelphia, PA, USA) [14Peterson E.J. Woods M.L. Dmowski S.A. Derimanov G. Jordan M.S. Wu J.N. Myung P.S. Liu Q.H. Pribila J.T. Freedman B.D. Shimizu Y. Koretzky G.A. Coupling of the TCR to integrin activation by Slap‐130/Fyb.Science. 2001; 293: 2263-5Crossref PubMed Scopus (267) Google Scholar]; GPVI‐deficient (GPVI−/−) C57/Bl6 mice were as previously reported [18Kato K. Kanaji T. Russell S. Kunicki T.J. Furihata K. Kanaji S. Marchese P. Reininger A. Ruggeri Z.M. Ware J. The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion.Blood. 2003; 102: 1701-7Crossref PubMed Scopus (236) Google Scholar]; WT C57/Bl6 mice were from Harlan (Bicester, UK); Ha1/29 (anti‐murine α2) and Ha31/8 (anti‐murine α1) antibodies were from BD Biosciences Pharmingen (Oxford, UK); fluorescein isothiocyanate (FITC)‐conjugated rat antibodies against murine αIIbβ3 (Leo.H4), α2 (Sam.G4) and GPVI (JAQ1), FITC‐conjugated rat IgG and non‐conjugated anti‐GPVI JAQ1 and Six.E10 were from EMFRET Analytics (Eibelstadt, Germany); Immulon‐2HB plates were from Fisher Scientific (Loughborough, UK); Horm collagen (fibrillar, equine tendon type I) was from Axis‐Shield (Dundee, UK); lotrafiban was from GlaxoSmithKline (King of Prussia, PR, USA); heparin (Monoparin) was from CP Pharmaceuticals (Wrexham, UK); other reagents were from Sigma (Poole, UK). Platelets were from adult mice. Blood was drawn under terminal anesthesia into heparin (5 IU mL−1) and citrate (11 mm). Platelet‐rich plasma (PRP) was obtained by centrifugation (110 × g, 5 min). PRP containing prostaglandin E1 (2 μm) was further centrifuged (2000 × g, 6 min). The platelet pellet was resuspended in Calcium Free Tyrode’s (CFT) (137 mm NaCl, 11.9 mm NaHCO3, 0.4 mm NaH2PO4, 2.7 mm KCl, 1.1 mm MgCl2, 5.6 mm glucose; pH 7.4). Flow cytometry was used to determine surface levels of GPVI, αIIbβ3, and α2. Five microliters of FITC‐labeled antibody were incubated with 25 μL of 0.5 × 106 mL−1 washed platelets for 15 min. Platelets were diluted with 400 μL of phosphate‐buffered saline, and the samples were analyzed with a Becton Dickinson FACSCalibur (Becton Dickinson, Oxford, UK). The synthesis of CRP, GPP, III‐04 and GFOGER and the determination of melting temperatures (Tm) by polarimetry have been described previously [19Jarvis G.E. Raynal N. Langford J.P. Onley D.J. Andrews A. Smethurst P.A. Farndale R.W. Identification of a major GpVI‐binding locus in human type III collagen.Blood. 2008; 111: 4986-96Crossref PubMed Scopus (58) Google Scholar, 20Raynal N. Hamaia S.W. Siljander P.R. Maddox B. Peachey A.R. Fernandez R. Foley L.J. Slatter D.A. Jarvis G.E. Farndale R.W. Use of synthetic peptides to locate novel integrin alpha2beta1‐binding motifs in human collagen III.J Biol Chem. 2006; 281: 3821-31Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 21Knight C.G. Morton L.F. Onley D.J. Peachey A.R. Ichinohe T. Okuma M. Farndale R.W. Barnes M.J. Collagen‐platelet interaction: Gly‐Pro‐Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.Cardiovasc Res. 1999; 41: 450-7Crossref PubMed Scopus (188) Google Scholar, 22Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. The collagen‐binding A‐domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple‐helical) collagens.J Biol Chem. 2000; 275: 35-40Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar]. Sequences and Tm values are shown in Table S1. CRP used in aggregation, thromboxane B2 (TxB2) and signaling studies was cross‐linked [19Jarvis G.E. Raynal N. Langford J.P. Onley D.J. Andrews A. Smethurst P.A. Farndale R.W. Identification of a major GpVI‐binding locus in human type III collagen.Blood. 2008; 111: 4986-96Crossref PubMed Scopus (58) Google Scholar]. Turbidimetric aggregometry was carried out as previously described [23Jarvis G.E. Platelet aggregation: turbidimetric measurements.Methods Mol Biol. 2004; 272: 65-76PubMed Google Scholar], with Bio Data PAP‐4 and Chronolog aggregometers. Aggregation was monitored at 37 °C for 6 min following addition of agonist, after which thromboxane A2 (TxA2) production was measured as its stable metabolite, TxB2, with an ELISA kit (Assay Designs, Ann Arbor, MI, USA). The platelet count was 2.0 × 108 mL−1 except for aggregation studies in the presence of cangrelor and ADP, when it was 1.5 × 108 mL−1. Signaling methods have been described elsewhere [24Pearce A.C. Senis Y.A. Billadeau D.D. Turner M. Watson S.P. Vigorito E. Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen.J Biol Chem. 2004; 279: 53955-62Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar]. Briefly, tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) and Syk was measured following immunoprecipitation by the use of SDS‐PAGE and western blotting with anti‐phosphotyrosine 4G10 (Millipore, Watford, UK). Protein bands on scanned images of autoradiographs were quantified with Adobe Photoshop CS. The ratio of the phosphorylation signal to the total protein signal was analyzed in a similar way to the xCELLigence data (see below). Platelet adhesion was measured by detecting acid phosphatase in platelet lysates [25Onley D.J. Knight C.G. Tuckwell D.S. Barnes M.J. Farndale R.W. Micromolar Ca2+ concentrations are essential for Mg2+‐dependent binding of collagen by the integrin alpha 2beta 1 in human platelets.J Biol Chem. 2000; 275: 24560-4Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. One hundred microliters of substrate (10 μg mL−1 in 0.01 m acetic acid) were added to 96‐well plates and incubated overnight at 4 °C. Excess ligand was discarded, and the wells were blocked with 175 μL of 5% bovine serum albumin (BSA) in CFT for 1–2 h. Plates were washed three times with 175 μL per well of 0.1% BSA/CFT. Platelets were pretreated with inhibitors 15 min before addition to the wells. Preactivation with U46619 was performed immediately before addition to the plate. Platelets (1.25 × 108 mL−1, 50 μL per well) were then incubated at room temperature for 1 h. Excess platelets were discarded, and the wells were washed three times. One hundred and fifty microliters of citrate lysis buffer (3.53 mmp‐nitrophenyl phosphate, 71.4 mm trisodium citrate, 28.55 mm citric acid, 0.1% [v/v] Triton X‐100; pH 5.4) were added for 1 h at room temperature. One hundred microliters of 2 m NaOH were added to each well, and absorbance at 405 nm was measured with a Fluostar Optima plate reader (BMG Labtech, Aylesbury, UK). The relationship between human platelet number and absorbance has been reported previously [25Onley D.J. Knight C.G. Tuckwell D.S. Barnes M.J. Farndale R.W. Micromolar Ca2+ concentrations are essential for Mg2+‐dependent binding of collagen by the integrin alpha 2beta 1 in human platelets.J Biol Chem. 2000; 275: 24560-4Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]: Fig. S1 shows similar data for mouse platelets. The xCELLigence system (Roche Diagnostics, Burgess Hill, UK) is a label‐free technology that quantifies cell adhesion and spreading in real time [26Castillo‐Briceno P. Bihan D. Nilges M. Hamaia S. Meseguer J. Garcia‐Ayala A. Farndale R.W. Mulero V. A role for specific collagen motifs during wound healing and inflammatory response of fibroblasts in the teleost fish gilthead seabream.Mol Immunol. 2011; 48: 826-34Crossref PubMed Scopus (40) Google Scholar, 27Ke N. Wang X. Xu X. Abassi Y.A. The xCELLigence system for real‐time and label‐free monitoring of cell viability.Methods Mol Biol. 2011; 740: 33-43Crossref PubMed Scopus (325) Google Scholar]. It measures electrical impedance across a pair of gold‐plated interdigitated microelectrodes on specialized 96‐well E‐plates (Fig. S2). Impedance is reported as a cell index value. E‐plates were substrate‐coated as for static adhesion. Fifty microliters of CFT were added to each well and allowed to equilibrate at 37 °C, and baseline impedance measurements were recorded. Fifty microliters of washed platelets (2.5 × 108 mL−1) were then added to give a final platelet count of 1.25 × 108 mL−1. Impedance was recorded every minute for 2.5 h. Fifty microliters of platelets (2 × 107 mL−1) were added to each well of an eight‐well glass slide, coated with the indicated peptide as for the static adhesion assay. The morphology of adherent and spreading platelets was determined by wide‐field differential‐interference contrast microscopy with the transmitted light facility of an Olympus IX‐81 FV300 confocal microscope (Olympus, Southend‐on‐Sea, UK) with a × 60 PlanApo objective and a numerical aperture of 1.42. Bone marrow was isolated from murine femora and tibiae. Megakaryocytes were cultured and purified, and migration and polyploidy analysis were carried out as described previously [28Mazharian A. Watson S.P. Severin S. Critical role for ERK1/2 in bone marrow and fetal liver‐derived primary megakaryocyte differentiation, motility, and proplatelet formation.Exp Hematol. 2009; 37: 1238-49Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]. Surface levels of GPIb, α2 and αIIb and apoptosis were determined by flow cytometry [29Tijssen M.R. Woelders H. De Vries‐van Rossen A. van der Schoot C.E. Voermans C. Lagerberg J.W. Improved postthaw viability and in vitro functionality of peripheral blood hematopoietic progenitor cells after cryopreservation with a theoretically optimized freezing curve.Transfusion. 2008; 48: 893-901Crossref PubMed Scopus (11) Google Scholar]. Spleens from mice of known age, sex and weight were removed, debrided, and weighed. Spleens were fixed in 3.6% formaldehyde, mounted, and stained with hematoxylin and eosin. Megakaryocytes were quantified in each spleen by counting their number in 10 randomly selected high‐power (× 60) fields. Aggregation data were fitted to four‐parameter logistic equations [30Jarvis G.E. Atkinson B.T. Frampton J. Watson S.P. Thrombin‐induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb.Br J Pharmacol. 2003; 138: 574-83Crossref PubMed Scopus (39) Google Scholar]. The effect of genotype on pA50 (‐log EC50) values was assessed with paired t‐tests and anova. Analysis of xCELLigence data was performed on integrated cell index values (0–2.5 h). Analysis of static adhesion data was performed on absorbance values transformed as previously described [19Jarvis G.E. Raynal N. Langford J.P. Onley D.J. Andrews A. Smethurst P.A. Farndale R.W. Identification of a major GpVI‐binding locus in human type III collagen.Blood. 2008; 111: 4986-96Crossref PubMed Scopus (58) Google Scholar], to minimize heteroscedasticity. Responses that were not significantly different from the negative controls, GPP and BSA, were excluded. Data were analysed by two‐way anova with treatment condition as a fixed factor and experimental date as a random factor. Treatment conditions were grouped into homogeneous subsets with the Waller–Duncan test (type I/type II error ratio = 100). Conditions within a subset were considered to be not significantly different. Logged spleen weights were analysed with ancova: sex and genotype were fixed factors; age and body weight were covariates. Analyses were carried out with Microsoft Excel 2007 and spss 16.0 for Windows (IBM, Portsmouth, UK). Flow cytometry was used to determine the levels of GPVI, α2 and αIIbβ3 on WT, ADAP−/− and FcRγ−/− platelets (Fig. S3). There were no differences in the levels of α2. The levels of GPVI were the same on WT and ADAP−/− platelets, and GPVI was undetectable on FcRγ−/− platelets. The level of αIIbβ3 was 5% lower on ADAP−/− platelets than on WT platelets (P < 0.05). The functional effect of this difference would be minor. Others have found no significant difference in αIIbβ3 levels [15Kasirer‐Friede A. Moran B. Nagrampa‐Orje J. Swanson K. Ruggeri Z.M. Schraven B. Neel B.G. Koretzky G. Shattil S.J. ADAP is required for normal alphaIIbbeta3 activation by VWF/GP Ib–IX–V and other agonists.Blood. 2007; 109: 1018-25Crossref PubMed Scopus (53) Google Scholar]. Aggregation of WT, ADAP−/− and FcRγ−/− platelets was induced by collagen, the GPVI‐selective agonist CRP, and the TP receptor agonist U46619 (Fig. 1). ADAP−/− platelets responded normally to CRP and U46619; however, there was a two‐fold rightward shift of the collagen concentration–response curves for rate and extent of aggregation (Table 1). The time to onset of collagen‐induced responses was extended in ADAP−/− platelets. In FcRγ−/− platelets, neither CRP nor collagen induced aggregation, although U46619‐induced aggregation was normal.Table 1EC50 values for the rate and extent of aggregation induced by collagen‐related peptide (CRP), collagen and U46619 in wild‐type (WT), adhesion and degranulation‐promoting adapter protein‐deficient (ADAP−/−) and Fc receptor γ‐chain‐deficient (FcRγ−/−) plateletsCRP (μg mL−1)Collagen (μg mL−1)U46619 (μm)Rate of aggregation: mean EC50 value (95% CI) WT (n = 9)1.1 (0.78–1.6)1.0 (0.83–1.2)0.43 (0.28–0.66) ADAP−/− (n = 9)1.4 (1.1–1.7)2.6 (1.7–3.9)0.68 (0.42–1.1) FcRγ−/− (n = 5)NRNR0.51 (0.27–0.94) P‐value0.21*< 0.0001*0.06†Extent of aggregation: mean EC50 value (95% CI) WT (n = 9)0.63 (0.39–1.0)0.89 (0.78–1.0)0.38 (0.25–0.57) ADAP−/− (n = 9)0.69 (0.44–1.1)2.1 (1.8–2.5)0.58 (0.37–0.93) FcRγ−/− (n = 5)NRNR0.45 (0.29–0.68) P‐value0.71*< 0.00001*0.01†CI, confidence interval; NR, not recorded. Statistical comparison of pA50 values (– log EC50) was carried out with paired t‐tests* or anova†. Open table in a new tab CI, confidence interval; NR, not recorded. Statistical comparison of pA50 values (– log EC50) was carried out with paired t‐tests* or anova†. TxB2 levels were determined 6 min after addition of agonist in the aggregation samples (Fig. 1). CRP‐induced and collagen‐induced TxB2 production were reduced by approximately 50% in ADAP−/− as compared with WT platelets, and were undetectable in FcRγ−/− platelets. U46619 induced no detectable TxB2 production. We investigated the possibility that reduced TxA2 synthesis was responsible for the reduction in collagen‐induced aggregation in ADAP−/− platelets. In the presence of aspirin (100 μm), there remained a significant reduction in collagen‐induced aggregation in ADAP−/− platelets (P < 0.01). CRP‐induced aggregation was the same in WT and ADAP−/− platelets in the absence and presence of aspirin (Fig. 2A,B). We further examined the effect of ADAP deficiency in the absence of the influence of released ADP and TxA2. ADP was inhibited with cangrelor (P2Y12 receptor antagonist, 1 μm) and adenosine 3′,5′‐diphosphate (A3P5P: P2Y1 receptor antagonist, 1 mm). TxA2 synthesis was blocked with aspirin (100 μm). A lower platelet count (1.5 × 108 mL−1) and higher concentrations of collagen (50 μg mL−1) and CRP (10 μg mL−1) were used to overcome the effects of the inhibitors. The response to collagen was significantly lower in ADAP−/− platelets. There was also a significant effect on CRP, although this was proportionately smaller than for collagen (Fig. 2C,D). CRP‐induced and collagen‐induced tyrosine phosphorylation of Syk and PLCγ2 were measured in WT and ADAP−/− platelets (Fig. 3). There was no change in the phosphorylation of Syk in ADAP−/− platelets. Phosphorylation of PLCγ2 induced by collagen (10 μg mL−1) was substantially attenuated in ADAP−/− platelets. A small reduction in PLCγ2 phosphorylation induced by CRP (10 μg mL−1) was not statistically significant (Fig. 3B). Platelet adhesion is dependent on direct interactions with ligands and secondary activation processes. These processes were investigated with collagen, CRP, GFOGER (α2β1‐selective ligand [22Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. The collagen‐binding A‐domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple‐helical) collagens.J Biol Chem. 2000; 275: 35-40Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar]), and III‐04, a peptide that supports α2β1‐dependent and GPVI‐dependent adhesion [19Jarvis G.E. Raynal N. Langford J.P. Onley D.J. Andrews A. Smethurst P.A. Farndale R.W. Identification of a major GpVI‐binding locus in human type III collagen.Blood. 2008; 111: 4986-96Crossref PubMed Scopus (58) Google Scholar] (Fig. 4). Adhesion to BSA and GPP (data not shown) was indistinguishable from that of FcRγ−/− platelets to CRP. Adhesion to CRP was unaffected by α2‐blocking or α1‐blocking antibodies, but was reduced by lotrafiban (αIIbβ3 antagonist) and EDTA. There was no significant effect of ADAP deficiency on platelet adhesion to CRP. Preactivation of lotrafiban‐treated platelets with U46619 did not increase binding to CRP. Adhesion to GFOGER was significantly reduced in both ADAP−/− and FcRγ−/− platelets. Lotrafiban reduced adhesion of WT but not ADAP−/− or FcRγ−/− platelets, suggesting that the greater binding of the WT platelets was attributable to αIIbβ3‐mediated interactions. Preactivation of lotrafiban‐treated platelets with U46619 increased binding of WT, ADAP−/− and FcRγ−/− platelets to similar levels as that of control WT platelets, indicating that ADAP and FcRγ participate in activatory signaling following adhesion to GFOGER. Adhesion of FcRγ−/− platelets to III‐04 was substantially reduced. Residual binding was blocked by the α2‐blocking antibody, showing that adhesion to III‐04 is dependent on both GPVI and α2β1. Adhesion of ADAP−/− platelets was also reduced. Lotrafiban attenuated binding of WT but not ADAP−/− or FcRγ−/− platelets. Adhesion of the three genotypes was the same for U46619‐activated lotrafiban‐treated platelets. Adhesion to collagen was reduced in FcRγ−/− platelets. The reduction in binding of ADAP−/− platelets was not statistically significant. Lotrafiban had no significant effect on binding. Preactivation of lotrafiban‐treated platelets with U46619 restored FcRγ−/− binding levels to that observed with WT and ADAP−/− platelets. In the presence of the α2‐blocking antibody, ADAP deficiency further reduced adhesion as compared with WT platelets. We have previously observed reduced adhesion of FcRγ−/− platelets to GFOGER [19Jarvis G.E. Raynal N. Langford J.P. Onley D.J. Andrews A. Smethurst P.A. Farndale R.W. Identification of a major GpVI‐binding locus in human type III collagen.Blood. 2008; 111: 4986-96Crossref PubMed Scopus (58) Google Scholar]. This suggests that FcRγ may contribute to α2β1 function or that GFOGER may interact with GPVI. To investigate this, we compared the adhesion of WT, GPVI−/− (which express FcRγ [18Kato K. Kanaji T. Russell S. Kunicki T.J. Furihata K. Kanaji S. Marchese P. Reininger A. Ruggeri Z.M. Ware J. The contribution of glycoprotein VI to stable platelet adhesion and thrombus formation illustrated by targeted gene deletion.Blood. 2003; 102: 1701-7Crossref PubMed Scopus (236) Google Scholar]) and FcRγ−/− (which" @default.
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